Diabetes is a group of diseases characterized by, among other features, defects in the regulation of glucose utilization and metabolism, resulting in impaired glucose tolerance. Despite the availability of insulin replacement therapy and a number of other therapeutic medications, which have reduced the acute mortality associated with diabetic ketoacidosis, insulin-treated patients inevitably develop long-term complications that may result in renal failure, loss of sight, as well as chronic and debilitating peripheral and cardiovascular disease. The major forms of diabetes include insulin-dependent diabetes mellitus, characterized by a deficiency of endogenous insulin secretion and non-insulin-dependent diabetes mellitus, characterized by a relative resistance of body tissues to circulating insulin. Both types of diabetes respond to administration of exogenous insulin. The various commercially available insulin preparations are protein materials that must be injected and that are associated with all of the other disadvantages that accompany the administration of foreign proteins to a patient. Previous efforts to provide oral therapeutic agents have resulted in oral hypoglycemic agents, such as the sulfonylureas, that are believed to act primarily by stimulating endogenous insulin secretion. Nevertheless, both the first and second generation sulfonylureas suffer from a number of drawbacks including hypoglycemia especially when associated with renal impairment and have been associated with a number of health risks, such as hypoglycemia and adverse effects in the cardiovascular and the central nervous system. In addition, cell replacement therapy harbors severe risks of transfer of infectious agents.
It is known that pancreatic xcex2-cells contain several cyclic nucleotide phosphodiesterases that can be activated under different physiological conditions to lower the levels of cyclic AMP and reduce insulin secretion. Thus, it has been thought that inhibition of cyclic nucleotide phosphodiesterases of pancreatic xcex2-cells would be a potentially powerful approach to enhancing insulin secretion in a glucose dependent fashion which also circumvents the development of the adverse effects of hypoglycemia. Pancreatic xcex2-cells contain several cyclic nucleotide phosphodiesterases that can be activated under different physiological conditions to lower the levels of cyclic AMP and reduce insulin secretion.
For this reason, attempts have been made to identify those nucleotide phosphodiesterases that function in concert with glucose to limit insulin secretion, and which lack a strong requirement for additional hormonal or neural stimulation. Identification of such cyclic nucleotide phosphodiesterases would also provide valuable targets for the development of novel anti-hyperglycemic agents. However, to date, previous efforts to identify pancreatic xcex2-cell phosphodiesterases relevant to glucose dependent insulin secretion have been unsuccessful. Contradictory results have been reported by several studies that implicate PDE3 in regulation of glucose dependent insulin secretion (Shafiee-Nick et al., Br J Pharmacol. 115:1486-92, 1995; Parker et al., Biochem Biophys Res Commun. 217:916-23, 1995; Leibowitz et al., Diabetes 44:67-74, 1995; Zhao et al., Proc Natl Acad Sci USA 94: 3223-8, 1997). Data concerning PDE4 is controversial (Shafiee-Nick et al., Br J Pharmacol. 115:1486-92, 1995; Parker et al., Biochem Biophys Res Commun. 217:916-23, 1995; Leibowitz et al., Diabetes 44:67-74, 1995; Zhao et al., Proc Natl Acad Sci USA 94: 3223-8, 1997). However, more current studies demonstrate effects of PDE3 only in the presence of hormone regulators like insulin like growth factor 1 and leptin (Zhao et al., Proc Natl Acad Sci USA 94:3223-8, 1997; Zhao et al., J.Clin.Invest. 102:869-872, 1998). Further, these studies show that PDE4 does not affect insulin secretion under these circumstances (Zhao et al., Proc Natl Acad Sci USA 94:3223-8, 1997; Zhao et al., J.Clin.Invest. 102:869-872, 1998). In addition, the presence of PDE3 in adipocytes and in liver and its contribution to insulin action in these tissues, make that enzyme an unsuitable target for the treatment of hyperglycemia. Accordingly, in vivo administration of PDE3 inhibitors to rats failed to affect fasting or post-glucose plasma glucose levels (El-Metwally et al., Eur J Pharmacol. 324:227-32, 1997, Parker et al. Biochem Biophys Res Commun. 236:665-9, 1997).
Complications associated with insulin administration involve the introduction of foreign proteins to patients, and with cell replacement therapy the introduction of infectious agents. Complications associated with oral hypoglycemia agents involve the uncoupling of insulin secretion from nutritional, hormonal and neural regulation, hypoglycemia and other adverse effects. For these reasons, there remains a need in the art for new agents useful in the treatment of the various types of diabetes and for new methods of identifying such agents.
Pancreatic xcex2-cells contain multiple cyclic nucleotide phosphodiesterases that lower cAMP levels and reduce insulin secretion. Inhibition of xcex2-cell cAMP phosphodiesterases can augment insulin secretion in a nutrient, hormone and neural sensitive fashion, and thus provide a powerful approach for regulating or increasing insulin secretion. Thus far, xcex2-cell cyclic nucleotide phosphodiesterases that can serve as targets for regulating or increasing insulin secretion were not identified (Shafiee-Nick et al., Br. J. Pharmacol. 115:1486-92, 1995; Parker et al., Biochem. Biophys. Res. Commun. 217:916-23, 1995; Leibowitz et al., Diabetes 44:67-74, 1995; Zhao et al., Proc. Natl. Acad. Sci. USA 94: 3223-8, 1997; Zhao et al., J. Clin. Invest. 102:869-872, 1998; El-Metwally et al., Eur. J. Pharmacol. 324:227-32, 1997, Parker et al., Biochem. Biophys. Res. Commun. 236:665-9, 1997.
The second messengers cAMP and cGMP mediate diverse physiological responses to hormones, neurotransmitters and light. Rates of cyclic nucleotide synthesis by cyclases and of their degradation by phosphodiesterases (PDEs) regulate their cellular concentrations (reviewed in Beavo, J. A. (1995) Physiol. Rev. 75, 725-748 and Houslay, M. D. and Milligan, G. (1997) TIBS 217-224). Cyclic nucleotide PDEs have been distinguished into nine families based on their substrate affinity and specificity, their selective sensitivity to cofactors and inhibitory drugs. Cyclic nucleotide PDE families are: (1) PDE1xe2x80x94Ca+2/calmodulin stimulated PDEs; (2) PDE2xe2x80x94cGMP stimulated PDEs; (3) PDE3xe2x80x94cGMP inhibited PDEs; (4) PDE4xe2x80x94cAMP specific PDEs; (5) PDE5xe2x80x94cGMP specific PDEs; (6) PDE6xe2x80x94photoreceptor PDEs; and (7) PDE7xe2x80x94higher affinity cAMP specific PDEs; (8) PDE8xe2x80x94cAMP specific IBMX resistant PDEs (Fisher, et al. (1998) Biochem. Biophys. Res. Commun. 246, 570-577; Hayashi, et al. (1998) Biochem. Biophys. Res. Commun. 250, 751-756; Soderling, et al. (1998) Proc. Natl. Acad. Sci. USA 95, 8991-8996); (9) PDE9xe2x80x94cGMP specific IBMX resistant PDEs (Fisher, et al. (1998) J. Biol. Chem. 273, 15559-15564 and Soderling, et al. (1998) J. Biol. Chem. 273, 15553-15558). All mammalian PDEs contain a related C-terminal domain with xcx9c30% sequence identity between families, and N-terminal regulatory domains containing cofactor or cGMP binding sites, localization and other regulatory sequences. Both tissue and cell specific gene expression, and a variable splicing pattern, contribute to the unique and complex composition of cyclic nucleotide PDEs in mammalian cells normally containing activities derived from several families of PDEs (Beavo, J. A. (1995) Physiol. Rev. 75, 725-748 and Houslay, M. D. and Milligan, G. (1997) TIBS 217-224). PDE inhibitors that do not affect adenosine uptake and exhibit high selectivity between PDE families, and in some cases between PDE isozymes, are powerful tools for identification of PDEs involved in diverse physiological responses (Ballard, et al. (1998) J Urol 159, 2164-2171; Giembycz, et al. (1996) J. Pharmocology 118, 1945-1958; Zhao, et al. (1997) Proc. Natl. Acad. Sci. USA 94, 3223-3228).
Insulin secretion from pancreatic xcex2-cells is governed by the interplay between nutritional secretagogues and regulatory hormonal and neural stimuli (Rasmussen, et al. (1990) Diabetes 13, 655-665; Holz, G. G. and Habener, J. F. (1992) Trends in Biochem. Sci. 17, 388-393; Liang, Y. and Matschinsky, F. M. (1994) Annu. Rev. Nutr. 14, 59-81). Glucose, the major insulin secretagogue, triggers insulin release through calcium dependent vesicular exocytosis (Rasmussen, et al. (1990) Diabetes 13, 655-665; Holz, G. G. and Habener, J. F. (1992) Trends in Biochem. Sci. 17, 388-393; Liang, Y. and Matschinsky, F. M. (1994) Annu. Rev. Nutr. 14, 59-81; Ashcroft, S. J. and Ashcroft, F. M. (1992) Insulin: Molecular Biology to Pathology, Oxford Univ. Press, New York; German, M. S. (1993) Proc. Natl. Acad. Sci. USA 90, 1781-1785; Newgard, C. B. and McGarry, J. D. (1995) Annu. Rev. Biochem. 64, 689-719; Efrat, et al. (1994) Trends in Biochem. Sci. 19, 535-538). The glucose signal cascade leads both to membrane depolarization and a calcium influx via to the opening of L-type voltage sensitive calcium channels, and to other effects that include release of calcium from intracellular stores (Liang, Y. and Matschinsky, F. M. (1994) Annu. Rev. Nutr. 14, 59-81; Takasawa, et al. (1998) J. Biol. Chem. 273, 2497-2500; and Kajimoto, et al. (1996) Biochem. Biophys. Res. Commun. 219, 941-946). Hormones and insulinotropic gut factors that stimulate cAMP synthesis strongly augment glucose induced insulin secretion (Rasmussen, et al. (1990) Diabetes 13, 655-665; Holz, G. G. and Habener, J. F. (1992) Trends in Biochem. Sci. 17, 388-393; Liang, Y. and Matschinsky, F. M. (1994) Annu. Rev. Nutr. 14, 59-81; and Ashcroft, S. J. and Ashcroft, F. M. (1992) Insulin: Molecular Biology to Pathology, Oxford Univ. Press, New York). Conversely, hormonal inhibition of insulin secretion involves reductions in cAMP levels (Zhao, et al. (1997) Proc. Natl. Acad. Sci. USA 94, 3223-3228; D""Ambra, et al. (1990) Endocrinology 126, 2815-2822; Ma, et al. (1994) Endocrinology 134, 42-47; Grodsky, G. M. and Bolaffi, J. L. (1992) J. Cell Biochem. 48, 3-11; Bolaffi, et al. (1990) Endocrinology 126, 1750-1755). In addition to the role cAMP plays in hormonal modulation of insulin secretion, basal cAMP levels appear to be required for glucose to induce insulin secretion (Serre, et al. (1998) Endocrinology 139, 4448-4454). Potentiation of glucose induced insulin secretion is evident not only upon treatment of insulin secreting cells with the insulinotropic gut factor GLP1 (glucagon-like peptide 1), but also upon treatment with reagents that stimulate cAMP signaling including membrane permeable cAMP analogs, activators of adenyl cyclase, and PDE inhibitors (Rasmussen, et al. (1990) Diabetes 13, 655-665, D""Ambra, et al. (1990) Endocrinology 126, 2815-2822; Henquin, J. C. and Meissner, H. P. (1984) Endocrinology 115, 1125-1134; and Holz, G. G., Leech, C. A., and Habener, J. F. (1995) J. Biol. Chem. 270, 17749-17757). Like GLP1, these cAMP elevating agents do not induce significant insulin secretion in the absence of glucose. Targets for cAMP action are PKA substrates such as the voltage sensitive calcium channel, GLUT2 and potentially also ion channels to which cAMP binds directly (Liang, Y. and Matschinsky, F. M. (1994) Annu. Rev. Nutr. 14, 59-81; Leiser, M. and Fleischer, N. (1996) Diabetes 45, 1412-1418; Rajan, et al. (1989) Diabetes 38, 874-880; Ammala, et al. (1993) Nature 363, 356-358; Thorens, et al. (1996) J. Biol. Chem. 271, 8075-8081). In addition to the potentiation of glucose and calcium dependent insulin secretion, cAMP-stimulated exocytosis via calcium independent mechanisms is evident in patch clamped cells, and the contribution of this mechanism to insulin secretion under physiological conditions remains to be determined (Leiser, M. and Fleischer, N. (1996) Diabetes 45, 1412-1418; and Ammala, C., Ashcroft, F. M., and Rorsman, P. (1993) Nature 363, 356-358). A requirement for the localization of PKA to specific sites within pancreatic xcex2-cells via anchor proteins has been demonstrated for GLP-1 potentiation of insulin secretion (Lester, L. B., Langerberg, L. K., and Scott, J. D. (1997) Proc. Natl. Acad. Sci. USA 94, 14942-14947).
The involvement of cyclic nucleotide PDEs in the regulation of insulin secretion is inferred from the stimulatory effects of the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) on insulin secretion from insulin secreting cell lines, from islets, and from transgenic mice expressing a constitutively activated Gsxcex1 mutant in their pancreatic xcex2-cells (Rasmussen, et al. (1990) Diabetes 13, 655-665; D""Ambra, et al. (1990) Endocrinology 126, 2815-2822; Ma, et al. (1994) Endocrinology 134, 42-47; Henquin, J. C. and Meissner, H. P. (1984) Endocrinology 115, 1125-1134). Cyclic nucleotide PDEs present in xcex2-cells were thus far investigated as total PDE activities of crude islet extracts and the presence of PDEs 3 and 4, and calcium sensitive PDEs, in xcex2-cells has been inferred from these studies (Henquin, J. C. and Meissner, H. P. (1984) Endocrinology 115, 1125-1134). The involvement of PDE3 in glucose induced insulin secretion from pancreatic islets has been demonstrated in studies using selective PDE3 inhibitors (Henquin, J. C. and Meissner, H. P. (1984) Endocrinology 115, 1125-1134; Lipson, L. G. and Oldham, S. B. (1983) Life Sci 32, 775-780; Leibowitz, et al. (1995) Diabetes 46, 67-74; El-Metwally, M., Shafiee-Nick, et al. (1997) Eur. J. Pharmacol. 324, 227-232). The presence of PDE3B in pancreatic xcex2-cells and its involvement in IGF-1 and in leptin mediated inhibition of insulin secretion has been demonstrated recently (Zhao, et al. (1997) Proc. Natl. Acad. Sci.USA 94, 3223-3228; and Zhao, A. Z., Bornfeldt, K. E., and Beavo, J. A. (1998) J. Clin. Invest. 102, 869-873). However, in cultured pancreatic xcex2-cells PDE3B does not appear to play a role in insulin secretion induced by glucose in the absence of hormone regulation (Zhao, A. Z., Zhao, H., Teague, J., Fujimoto, W., and Beavo, J. A. (1997) Proc. Natl. Acad. Sci. USA 94, 3223-3228; and Zhao, A. Z., Bornfeldt, K. E., and Beavo, J. A. (1998) J. Clin. Invest. 102, 869-873).
The present invention provides for a method of identifying novel agents that increase glucose dependent insulin secretion in pancreatic islet cells as well as methods of treating diabetes using agents which have an inhibitory effect on the activity of pancreatic islet cell phosphodiesterases (xe2x80x9cPDExe2x80x9d) enzyme, namely PDE1C. The methods described herein are based upon the inventor""s surprising discovery that inhibition of PDE1C increases glucose dependent insulin secretion.
Specifically, the present invention provides for a method of identifying therapeutic agents that act to regulate or increase the release of insulin from pancreatic islet cells. The method of identification provided herein is used to determine the effects of isozyme specific phosphodiesterase inhibitors on insulin secretion from cultured pancreatic xcex2-cells.
Further, the present invention provides for agents that have an inhibitory effect on the activity of PDE1C in pancreatic cells. Useful compositions according to the invention include, for example, compounds of the general formula: 3-isobutyl-1-methylxanthine derivatives with substitutions at positions 2 (R1) and 8 (R2). Preferably, R1 and R2 are independently alkyl (C1 to C3), fluoroalkyl (F1 to F3), chloroalkyl (Cl1 to Cl3), aryl (C5 to C6), fluoroaryl (F1 to F2), chloroaryl (Cl1 to Cl2).
Also provided by the present invention is a method of treating diabetes comprising administering to a subject an amount of a PDE1C inhibitor effective to treat the type II diabetes. The inhibitor may be selected from, for example, eburnamenine-14-carboxylic acid ethyl ester (vinpocetine), 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine (8MM-IBMX), zaprinast (MandB 22948), 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone (rolipram), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724), 1,6-dihydro-2-methyl-6-oxo-(3,4xe2x80x2-bipyridine)-5-carbonitrile (milrinone), trequinsin (HL 725), and/or combinations thereof.
Additional objects of the present invention will be apparent from the description which follows.